Effect of lipid-free apoE and apoAI on cholesterol efflux.

<p>(A–B) RAW 264.7 macrophages were incubated at 37°C for 48 h with 1 µCi/ml of ³H-cholesterol and 20 µg/ml apoE-free (E⁻) lipoprotein, followed by incubation with the indicated amount of apoE3 (E3) or apoE4 (E4) for an additional 5 h. Thereafter, cells were incubated with 20 µg/ml of apoAI or base medium alone for 2 h. Cholesterol efflux was determined as described in the Materials and Methods. ApoAI mediated cholesterol efflux was calculated as the total efflux in the presence of apoAI minus the efflux to the base medium without apoAI. (C) The cells were loaded with ³H-cholesterol as abovementioned, and then incubated with 0.44 µM of lipid free apoA1 (12.35 µg/ml), apoE3 or apoE4 (15 µg/ml), or culture medium alone as a control (ctrl) for 6 h. Total cholesterol efflux was determined as described in the Materials and Methods. Data represent the mean ± SEM from 3–4 separate experiments. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; <b>^#</b><i>P</i><0.05 relative to apoE4.</p

Effect of lipid-free apoE isoforms on Sp1 expression and phosphorylation.

<p>(A–D) RAW 264.7 macrophages were incubated at 37°C for 5 h with 3 µg/ml of lipid-free apoE3 (E3) or apoE4 (E4) or culture medium alone as a control (Ctrl). Sp1 protein was determined by immunoblotting. The level of phosphorylated Sp1 (top band) was expressed relative to the total Sp1 protein level (the sum of the top and bottom bands relative to actin) or actin. Data represent the mean ± SEM from 3–4 separate experiments. *P<0.05 or **P<0.01 relative to controls; <b>^#</b>P<0.05 relative to apoE3.</p

Effect of apoE enrichment on lipoprotein cholesterol profile.

<p>Fifty µg of apoE-free (E⁻) lipoproteins (<1.063) were incubated with 5 µg of apoE3 or apoE4 or an equivalent volume of vehicle at RT for 1 h. Cholesterol profiles in E^- lipoproteins (A), apoE3- (B), and apoE4-enriched (C) lipoproteins were analyzed with fast protein liquid chromatography.</p

Effect of lipid-associated apoE isoforms on Sp1 expression and phosphorylation.

<p>(A) RAW 264.7 cell lysates were incubated for 5 h with or without λ-phosphatase and Sp1 levels determined by immunoblotting. The upper band (p-Sp1) was determined to be the phosphorylated form (p-Sp1) and the lower band (unp-Sp1), the unphosphorylated form. (B–E) RAW 264.7 macrophages were incubated at 37°C for 5 h with 20 µg/ml of apoE-free (E⁻) lipoprotein, 20 µg/ml of E⁻ lipoproteins containing 2 µg/ml of apoE3 (E3) or apoE4 (E4) or culture medium alone (Ctrl). The level of phosphorylated Sp1 (top band) was expressed relative to the total Sp1 protein level (the sum of the top and bottom bands relative to actin) or actin. Data represent the mean ± SEM from 3–4 separate experiments. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; ^†<i>P</i><0.05 relative to E⁻; <b>^#</b><i>P</i><0.05 relative to apoE3.</p

Concentration-dependent effect of lipoprotein-associated apoE isoforms on ABCA1 expression.

<p>(A–B) RAW 264.7 macrophages were incubated for 5 h at 37°C with 20 µg/ml of apoE-free (E⁻) lipoprotein, 20 µg/ml of E⁻ lipoproteins carrying the indicated concentrations of apoE3 (E3) or apoE4 (E4), or with culture medium alone as a control (Ctrl). ABCA1 protein levels were determined by immunoblotting, and normalized relative to β-actin. (C) RAW 264.7 macrophages were incubated for 5 h at 37°C with 20 µg/ml of apoE-free (E^-) lipoprotein, 20 µg/ml of lipoproteins carrying 2 µg/ml of apoE3 (E3) or apoE4 (E4), or culture medium alone (Ctrl). ABCA1 mRNA levels were determined by quantitative real-time RT-PCR and normalized relative to GAPDH mRNA. Data represent the mean ± SEM from four separate experiments. ^†<i>P</i><0.05 relative to untreated controls; *<i>P</i><0.05 or **<i>P</i><0.01 relative to cells treated with E^- lipoprotein; <b>^#</b><i>P</i><0.05 relative to cells treated with apoE3-carrying lipoproteins.</p

Effect of lipid-free apoE isoforms on phosphorylation of PKCζ and Sp1, and ABCA1 expression in human THP-1 macrophages.

<p>(A–D) Human macrophage THP-1 cells were treated with 3 µg/ml of lipid-free apoEs for as described under Materials and Methods and the protein levels of ABCA1, p-PKCζ and Sp1 determined by immunoblotting. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; ^#<i>P</i><0.05 relative to apoE3.</p

Effect of lipoprotein-associated apoE isoforms on PI3K and PKCζ phosphorylation.

<p>RAW 264.7 macrophages were incubated at 37°C for 5 h with 20 µg/ml of apoE-free (E⁻) lipoprotein, 20 µg/ml of E⁻ lipoproteins containing 2 µg/ml of apoE3 (E3) or apoE4 (E4) or culture medium alone as a control (Ctrl). (A–C) Phosphorylated PI3K and PKCζ were determined by immunoblotting. Data represent the mean ± SEM from 3–4 separate experiments. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; ^†<i>P</i><0.05 relative to E⁻; <b>^#</b><i>P</i><0.05 relative to apoE3.</p

Effect of lipid-free apoE isoforms on PI3K and PKCζ phosphorylations.

<p>RAW 264.7 macrophages were incubated at 37°C for 5 h with 3 µg/ml of lipid-free apoE3 (E3) or apoE4 (E4) or culture medium alone as a control (Ctrl). (A–C) Phosphorylated PI3K and PKCζ were determined by immunoblotting. Data represent the mean ± SEM from 4 separate experiments. *<i>P</i><0.05 or **<i>P</i><0.01 relative to controls; <b>^#</b><i>P</i><0.05 relative to apoE3.</p

Effect of lipid-free apoE isoforms on the cholesterol level of lipid-laden macrophages.

<p>RAW264.7 macrophages were incubated at 37°C for 87 h with 20 µg/ml of apoE-free (E⁻) lipoproteins, followed by an additional 6h incubation with culture medium alone (E⁻/87 h), 3 µg/ml of lipid-free apoE3 (E⁻/87 h + E3), or apoE4 (E⁻/87 h + E4). Cells without lipoprotein and apoE treatments were used as controls (Ctrl). (A–B) Cellular total and free cholesterols were measured colorimetrically and esterified cholesterol was calculated from the difference between total and free cholesterol. Data represent the mean ± SEM from three independent experiments. **<i>P</i><0.01 relative to control; ^†<i>P</i><0.05 or^††<i>P</i><0.01 relative to E⁻/87 h; <b>^#</b><i>P</i><0.05 relative to E⁻/87 h + E3.</p